Letcher was editor of the (Shenandoah) Valley Star newspaper from 1840 to 1850. He was active in the presidential campaigns of 1840, 1844, and 1848, serving as Democratic elector in 1848. Although never a true abolitionist, he signed the Ruffner Pamphlet of 1847, which proposed the abolition of slavery in that part of Virginia west of the Blue Ridge Mountains; however, he soon repudiated this antislavery stand. He was a delegate to the Virginia Constitutional Convention of 1850.

He was elected as a Democratic candidate and served as a Representative in the United States Congress from 1851 to 1859. In Congress, he was known as "Honest John" because of his opposition to government extravagance.

John Letcher was elected as Governor of Virginia in 1859, defeating Whig candidate William L. Goggin, and served from 1860–1864. Letcher was prominent in the organization of the peace convention that met in Washington, D.C., February 8, 1861, in an effort to devise means to prevent the impending American Civil War. He discouraged secession, but was active in sustaining the ordinance passed by Virginia on April 17, 1861. Despite scheduling a popular vote to ultimately determine whether Virginia would secede from the Union, the actions of the Virginia Secession Convention and of the state government, especially Virginia Governor Letcher effectively took Virginia out of the Union.[1][2] The referendum occurred on May 23, 1861, and Virginia voters overwhelmingly approved the Articles of Secession.[3] Governor Letcher appointed Robert E. Lee, who had just resigned as a colonel in the U.S. Army, as commander in chief of Virginia’s army and navy forces on April 22, 1861 at the grade of major general.[4] On April 24, 1861, Virginia and the Confederate States agreed that the Virginia forces would be under the overall direction of the Confederate President, Jefferson Davis, pending completion of the process of Virginia joining the Confederate States.[4]

The tomb of John Letcher at Stonewall Jackson Memorial Cemetery (Lexington)

After the Civil War, Letcher resumed the practice of law in Lexington. He was elected as a member of the House of Delegates in the Virginia General Assembly 1875–1877. He was a member of the Board of Visitors of the Virginia Military Institute (VMI) 1866–1880 and served as president of the Board for ten years.

Letcher's son, John Davidson Letcher, was a professor at Oregon State University, serving as acting president from January 1892 to June 1892.
Governor Letcher had a daughter, Lizzie, who married James Harrison, a language professor at Washington and Lee and later head of the Romance and Teutonic Language Department at the University of Virginia after 1895.